Thrombopoetin receptor activator and process for producing the same

ABSTRACT

A preventive, therapeutic or improving agent for diseases against which activation of the thrombopoietin receptor is effective or a platelet increasing agent, which contains a thrombopoietin receptor activator represented by the formula (1): 
                         
[wherein each of R 1  and R 3  is independently a hydrogen atom, SO 3 H, a C 1-6  alkyl group, a C 1-6  alkylcarbonyl group or a C 6-18  arylcarbonyl group (the C 1-6  alkyl group, the C 1-6  alkylcarbonyl group and the C 6-18  arylcarbonyl group may be optionally substituted with a halogen atom, a hydroxyl group, a C 2-6  alkenyl group, a C 1-6  alkoxy group, a C 1-6  alkoxycarbonyl group, a C 6-18  aryl group, a 2-pyridyl group, a 3-pyridyl group, a 4-pyridyl group, a 2-furanyl group, a 3-furanyl group, a 2-thienyl group, a 3-thienyl group or NR 9 R 10 ) , and each of R 2 , R 4  and R a  is independently a hydrogen atom, a hydroxyl group or a C 1-6  alkoxy group].

TECHNICAL FIELD

The present invention relates to preventive, therapeutic and improvingagents having affinity for and agonistic action on the thrombopoietinreceptor for diseases against which activation of the thrombopoietinreceptor is effective. Specifically, it relates to pharmaceuticalcompositions comprising compounds which increase platelets throughstimulation of differentiation and proliferation of hematopoietic stemcells, megakaryocytic progenitor cells and megakaryocytes or compoundsused for therapeutic angiogenesis or with anti-arteriosclerosis actionthat stimulate differentiation and proliferation of vascular endothelialcells and endothelial progenitor cells.

BACKGROUND ART

Thrombopoietin is a cytokine consisting of 332 amino acids thatincreases platelet production by stimulating differentiation andproliferation of hematopoietic stem cells, megakaryocytic progenitorcells and megakaryocytes mediated by its receptor and therefore ispromising as a drug for hematological disorders. Recent reports that itstimulates differentiation and proliferation of vascular endothelialcells and endothelial progenitor cells have raised expectations oftherapeutic angiogenesis, anti-arteriosclerosis and prevention ofcardiovascular events (Microvasc. Res., 1999:58, 108-113, Circ. Res.,1999:84, 785-796, Blood 2001:98, 71a). Biologically active substanceswhich have been known so far to regulate platelet production through thethrombopoietin receptor include, in addition to thrombopoietin itself,the low molecular weight peptides having affinity for the thrombopoietinreceptor disclosed in JP-A-10-72492 (WO96/40750), WO96/40189, WO98/25965and the like. As a result of search for nonpeptidic low molecular weightcompounds that increase platelet production mediated by thethrombopoietin receptor, several reports have been made on low molecularweight compounds having affinity for the thrombopoietin receptor.

-   1) Applications filed by Hokuriku Seiyaku Co., Ltd. relating to    1,4-benzodiazepine derivatives (JP-A-11-1477 and JP-A-11-152276)-   2) International Laid-open Patent Applications filed by Shionogi &    Co., Ltd. (WO01/07423, WO01/53267, WO02/059099 and WO02/059100)-   3) International Laid-open Patent Applications filed by SmithKline    Beecham Corp (WO00/35446, WO00/66112, WO01/34585, WO01/17349,    WO01/39773, WO01/21180, WO01/89457, WO02/49413 and WO02/085343)-   4) Japanese Laid-open Patent Application filed by Torii    Pharmaceutical Co., Ltd. (JP-A-2001-97948)-   5) International Laid-open Patent Application filed by Roche    Diagnostics GMBH (WO99/11262)-   6) International Laid-open Patent Application filed by Yamanouchi    Pharmaceutical Co., Ltd. (WO02/06275)

Meanwhile, xanthocillin analogues are not only known to have broadantibacterial spectra but also reported to have antiviral action,aromatase inhibitory action (JP-A-07-69883), antitumor medicine(JP-A-02-304058 and JP-A-04-182427), anthelmintic agent (JP-A-02-40324),prostaglandin synthesis inhibitory action and platelet aggregationinhibitory action. However, nothing has been reported on affinity for oragonistic action on the thrombopoietin receptor.

Thrombopoietin and low molecular weight peptides having affinity for thethrombopoietin receptor are likely to be easily degraded in thegastrointestinal tract and are usually difficult to orally administer.As to thrombopoietin itself, the appearance of anti-thrombopoietinantibodies have been reported.

Besides, though it is probably possible to orally administer nonpeptidiclow molecular weight compounds, no practical drugs have been put on themarket.

Therefore, orally administrable low molecular weight compounds havingexcellent affinity for and agonistic action on the thrombopoietinreceptor as preventive, therapeutic and improving agents for diseasesagainst which activation of the thrombopoietin receptor is effectivehave been demanded. Specifically, low molecular weight compounds whichcan serve as platelet increasing agents or increasing agents for otherblood cells by stimulating differentiation and proliferation ofhematopoietic stem cells, megakaryocytic progenitor cells andmegakaryocytes or low molecular weight compounds which can be used fortherapeutic angiogenesis or as preventive and therapeutic agents forarteriosclerosis by stimulating endothelial cells and endothelialprogenitor cells have been demanded.

DISCLOSURE OF THE INVENTION

The present inventors conducted extensive research to find low molecularweight compounds having affinity for and agonistic action on thethrombopoietin receptor, and as a result, unexpectedly found thatconventionally known xanthocillin and its analogues and theirderivatives have high affinity for and agonistic action on thethrombopoietin receptor and found a novel process for producingxanthocillin analogues. The present invention was accomplished on thebasis of this discovery.

Namely, the present invention relates to a thrombopoietin receptoractivator represented by the formula (1)

[wherein each of R¹ and R³ is independently a hydrogen atom, SO₃H, aC₁₋₆ alkyl group, a C₁₋₆ alkylcarbonyl group or a C₆₋₁₈ arylcarbonylgroup (the C₁₋₆ alkyl group, the C₁₋₆ alkylcarbonyl group and the C₆₋₁₈arylcarbonyl group may be optionally substituted with a halogen atom, ahydroxyl group, a C₂₋₆ alkenyl group, a C₁₋₆ alkoxy group, a C₁₋₆alkoxycarbonyl group, a C₆₋₁₈ aryl group, a 2-pyridyl group, a 3-pyridylgroup, a 4-pyridyl group, a 2-furanyl group, a 3-furanyl group, a2-thienyl group, a 3-thienyl group (the C₆₋₁₈ aryl group, the 2-pyridylgroup, the 3-pyridyl group, the 4-pyridyl group, the 2-furanyl group,the 3-furanyl group, the 2-thienyl group and the 3-thienyl group may beoptionally substituted with a halogen atom or a C₁₋₆ alkyl group) orNR⁹R¹⁰ (wherein each of R⁹ and R¹⁰ is independently a hydrogen atom or aC₁₋₆ alkyl group (the C₁₋₆ alkyl group may be optionally substitutedwith a C₆₋₁₈ aryl group) or R⁹ and R¹⁰ mean, together with each other,—(CH₂)_(n)X(CH₂)_(m)— (wherein X is CR¹¹R¹² (wherein each of R¹¹ and R¹²is independently a hydrogen atom or a C₁₋₆ alkyl group (the C₁₋₆ alkylgroup may be optionally substituted with a C₆₋₁₈ aryl group)), NR¹³(wherein R¹³ is a hydrogen atom or a C₁₋₆ alkyl group (the C₁₋₆ alkylgroup may be optionally substituted with a C₆₋₁₈ aryl group)), O or S, nis 1, 2 or 3, and m is 1, 2 or 3, provided that n+m is 3, 4 or 5))), andeach of R², R⁴ and R^(a) is independently a hydrogen atom, a hydroxylgroup or a C₁₋₆ alkoxy group], a preventive, therapeutic or improvingagent against which activation of the thrombopoietin receptor iseffective, which contains the thrombopoietin receptor activator or aprodrug, pharmaceutically acceptable salt or solvate thereof, as anactive ingredient, a platelet increasing agent containing thethrombopoietin receptor activator or a prodrug, pharmaceuticallyacceptable salt or solvate thereof, as an active ingredient, a processfor producing a compound represented by the formula (2), which comprisesincubating a microorganism belonging to the Basipetospora genus andisolating the compound from the culture medium

(wherein each of R⁵ and R⁷ is independently a hydrogen atom or a methylgroup, and each of R⁶, R⁸ and R^(b) is independently a hydrogen atom, ahydroxyl group or a methoxy group), Basipetospora sp. strain No. 1142which is deposited under accession number FERM P-18940 and a compoundrepresented by the formula (3).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Evaluation of the effect of the compound of the presentinvention (xanthocillin X monomethyl ether) on the proliferation ofUT7/EPO-mpl cells by the MTT assay.

FIG. 2: Evaluation of the effect of the compound of the presentinvention (xanthocillin X monomethyl ether) on the proliferation ofUT7/EPO cells by the MTT assay.

FIG. 3: Evaluation of the effect of the compound of the presentinvention (3-methoxyxanthocillin X dimethyl ether) on the proliferationof UT7/EPO-mpl cells by the WST assay.

FIG. 4: Evaluation of the effect of the compound of the presentinvention (3-methoxyxanthocillin X dimethyl ether) on the proliferationof UT7/EPO cells by the WST assay.

BEST MODE FOR CARRYING OUT THE INVENTION

Now, the present invention will be described in detail.

In the present invention, “n” denotes normal, “i” denotes iso, “s”denotes secondary, “t” denotes tertiary, “c” denotes cyclo, “o” denotesortho, “m” denotes meta, and “p” denotes para.

First, the terms in the respective substituents R¹, R², R³, R⁴ and R^(a)will be explained.

A C₁₋₆ alkyl group may be linear, branched or cyclic (a C₃₋₆ cycloalkylgroup), and methyl, ethyl, n-propyl, i-propyl, c-propyl, n-butyl,i-butyl, s-butyl, t-butyl, c-butyl, 1-methyl-c-propyl,2-methyl-c-propyl, n-pentyl, 1-methyl-n-butyl, 2-methyl-n-butyl,3-methyl-n-butyl, 1,1-dimethyl-n-propyl, 1,2-dimethyl-n-propyl,2,2-dimethyl-n-propyl, 1-ethyl-n-propyl, c-pentyl, 1-methyl-c-butyl,2-methyl-c-butyl, 3-methyl-c-butyl, 1,2-dimethyl-c-propyl,2,3-dimethyl-c-propyl, 1-ethyl-c-propyl, 2-ethyl-c-propyl, n-hexyl,1-methyl-n-pentyl, 2-methyl-n-pentyl, 3-methyl-n-pentyl,4-methyl-n-pentyl, 1,1-dimethyl-n-butyl, 1,2-dimethyl-n-butyl,1,3-dimethyl-n-butyl, 2,2-dimethyl-n-butyl, 2,3-dimethyl-n-butyl,3,3-dimethyl-n-butyl, 1-ethyl-n-butyl, 2-ethyl-n-butyl,1,1,2-trimethyl-n-propyl, 1,2,2-trimethyl-n-propyl,1-ethyl-1-methyl-n-propyl, 1-ethyl-2-methyl-n-propyl, c-hexyl,1-methyl-c-pentyl, 2-methyl-c-pentyl, 3-methyl-c-pentyl,1-ethyl-c-butyl, 2-ethyl-c-butyl, 3-ethyl-c-butyl, 1,2-dimethyl-c-butyl,1,3-dimethyl-c-butyl, 2,2-dimethyl-c-butyl, 2,3-dimethyl-c-butyl,2,4-dimethyl-c-butyl, 3,3-dimethyl-c-butyl, 1-n-propyl-c-propyl,2-n-propyl-c-propyl, 1-i-propyl-c-propyl, 2-i-propyl-c-propyl,1,2,2-trimethyl-c-propyl, 1,2,3-trimethyl-c-propyl,2,2,3-trimethyl-c-propyl, 1-ethyl-2-methyl-c-propyl,2-ethyl-1-methyl-c-propyl, 2-ethyl-2-methyl-c-propyl,2-ethyl-3-methyl-c-propyl and the like may be mentioned.

A C₁₋₆ alkylcarbonyl group may be linear, branched or cyclic (a C₃₋₆cycloalkylcarbonyl group), and methylcarbonyl, ethylcarbonyl,n-propylcarbonyl, i-propylcarbonyl, c-propylcarbonyl, n-butylcarbonyl,i-butylcarbonyl, s-butylcarbonyl, t-butylcarbonyl, c-butylcarbonyl,1-methyl-c-propylcarbonyl, 2-methyl-c-propylcarbonyl, n-pentylcarbonyl,1-methyl-n-butylcarbonyl, 2-methyl-n-butylcarbonyl,3-methyl-n-butylcarbonyl, 1,1-dimethyl-n-propylcarbonyl,1,2-dimethyl-n-propylcarbonyl, 2,2-dimethyl-n-propylcarbonyl,1-ethyl-n-propylcarbonyl, c-pentylcarbonyl, 1-methyl-c-butylcarbonyl,2-methyl-c-butylcarbonyl, 3-methyl-c-butylcarbonyl,1,2-dimethyl-c-propylcarbonyl, 2,3-dimethyl-c-propylcarbonyl,1-ethyl-c-propylcarbonyl, 2-ethyl-c-propylcarbonyl, n-hexylcarbonyl,1-methyl-n-pentylcarbonyl, 2-methyl-n-pentylcarbonyl,3-methyl-n-pentylcarbonyl, 4-methyl-n-pentylcarbonyl,1,1-dimethyl-n-butylcarbonyl, 1,2-dimethyl-n-butylcarbonyl,1,3-dimethyl-n-butylcarbonyl, 2,2-dimethyl-n-butylcarbonyl,2,3-dimethyl-n-butylcarbonyl, 3,3-dimethyl-n-butylcarbonyl,1-ethyl-n-butylcarbonyl, 2-ethyl-n-butylcarbonyl,1,1,2-trimethyl-n-propylcarbonyl, 1,2,2-trimethyl-n-propylcarbonyl,1-ethyl-1-methyl-n-propylcarbonyl, 1-ethyl-2-methyl-n-propylcarbonyl,c-hexylcarbonyl, 1-methyl-c-pentylcarbonyl, 2-methyl-c-pentylcarbonyl,3-methyl-c-pentylcarbonyl, 1-ethyl-c-butylcarbonyl,2-ethyl-c-butylcarbonyl, 3-ethyl-c-butylcarbonyl,1,2-dimethyl-c-butylcarbonyl, 1,3-dimethyl-c-butylcarbonyl,2,2-dimethyl-c-butylcarbonyl, 2,3-dimethyl-c-butylcarbonyl,2,4-dimethyl-c-butylcarbonyl, 3,3-dimethyl-c-butylcarbonyl,1-n-propyl-c-propylcarbonyl, 2-n-propyl-c-propylcarbonyl,1-i-propyl-c-propycarbonyl, 2-i-propyl-c-propylcarbonyl,1,2,2-trimethyl-c-propylcarbonyl, 1,2,3-trimethyl-c-propylcarbonyl,2,2,3-trimethyl-c-propylcarbonyl, 1-ethyl-2-methyl-c-propylcarbonyl,2-ethyl-1-methyl-c-propylcarbonyl, 2-ethyl-2-methyl-c-propylcarbonyl,2-ethyl-3-methyl-c-propylcarbonyl or the like may be mentioned.

As a C₆₋₁₈ arylcarbonyl group, a benzoyl group, a 1-indenylcarbonylgroup, a 2-indenylcarbonyl group, a 3-indenylcarbonyl group, a4-indenylcarbonyl group, a 5-indenylcarbonyl group, a 6-indenylcarbonylgroup, a 7-indenylcarbonyl group, an α-naphthylcarbonyl group, aβ-naphthylcarbonyl group, a 1-tetrahydronaphthylcarbonyl group, a2-tetrahydronaphthylcarbonyl group, a 5-tetrahydronaphthylcarbonylgroup, a 6-tetrahydronaphthylcarbonyl group, an o-biphenylylcarbonylgroup, a m-biphenylylcarbonyl group, a p-biphenylylcarbonyl group, a1-anthrylcarbonyl group, a 2-anthrylcarbonyl group, a 9-anthrylcarbonylgroup, a 1-phenanthrylcarbonyl group, a 2-phenanthrylcarbonyl group, a3-phenanthrylcarbonyl group, a 4-phenanthrylcarbonyl group, a9-phenanthrylcarbonyl group or the like may be mentioned.

As a C₆₋₁₈ aryl group, a phenyl group, a 1-indenyl group, a 2-indenylgroup, a 3-indenyl group, a 4-indenyl group, a 5-indenyl group, a6-indenyl group, a 7-indenyl group, an α-naphthyl group, a β-naphthylgroup, a 1-tetrahydronaphthyl group, a 2-tetrahydronaphthyl group, a5-tetrahydronaphthyl group, a 6-tetrahydronaphthyl group, ano-biphenylyl group, a m-biphenylyl group, a p-biphenylyl group, a1-anthryl group, a 2-anthryl group, a 9-anthryl group, a 1-phenanthrylgroup, a 2-phenanthryl group, a 3-phenanthryl group, a 4-phenanthrylgroup, a 9-phenanthryl group or the like may be mentioned.

As a halogen atom, a fluorine atom, a chlorine atom, a bromine atom oran iodine atom may be mentioned.

A C₂₋₆ alkenyl group may be linear, branched or cyclic (a C₃₋₆cycloalkenyl group), and ethenyl, 1-propenyl, 2-propenyl,1-methyl-1-ethenyl, 1-butenyl, 2-butenyl, 3-butenyl,2-methyl-1-propenyl, 2-methyl-2-propenyl, 1-ethylethenyl,1-methyl-1-propenyl, 1-methyl-2-propenyl, 1-pentenyl, 2-pentenyl,3-pentenyl, 4-pentenyl, 1-n-propylethenyl, 1-methyl-1-butenyl,1-methyl-2-butenyl, 1-methyl-3-butenyl, 2-ethyl-2-propenyl,2-methyl-1-butenyl, 2-methyl-2-butenyl, 2-methyl-3-butenyl,3-methyl-1-butenyl, 3-methyl-2-butenyl, 3-methyl-3-butenyl,1,1-dimethyl-2-propenyl, 1-i-propylethenyl, 1,2-dimethyl-1-propenyl,1,2-dimethyl-2-propenyl, 1-c-pentenyl, 2-c-pentenyl, 3-c-pentenyl,1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl,1-methyl-1-pentenyl, 1-methyl-2-pentenyl, 1-methyl-3-pentenyl,1-methyl-4-pentenyl, 1-n-butylethenyl, 2-methyl-1-pentenyl,2-methyl-2-pentenyl, 2-methyl-3-pentenyl, 2-methyl-4-pentenyl,2-n-propyl-2-propenyl, 3-methyl-1-pentenyl, 3-methyl-2-pentenyl,3-methyl-3-pentenyl, 3-methyl-4-pentenyl, 3-ethyl-3-butenyl,4-methyl-1-pentenyl, 4-methyl-2-pentenyl, 4-methyl-3-pentenyl,4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl,1,2-dimethyl-1-butenyl, 1,2-dimethyl-2-butenyl, 1,2-dimethyl-3-butenyl,1-methyl-2-ethyl-2-propenyl, 1-s-butylethenyl, 1,3-dimethyl-1-butenyl,1,3-dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl, 1-i-butylethenyl,2,2-dimethyl-3-butenyl, 2,3-dimethyl-1-butenyl, 2,3-dimethyl-2-butenyl,2,3-dimethyl-3-butenyl, 2-i-propyl-2-propenyl, 3,3-dimethyl-1-butenyl,1-ethyl-1-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl,1-n-propyl-1-propenyl, 1-n-propyl-2-propenyl, 2-ethyl-1-butenyl,2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl,1-t-butylethenyl, 1-methyl-1-ethyl-2-propenyl,1-ethyl-2-methyl-1-propenyl, 1-ethyl-2-methyl-2-propenyl,1-i-propyl-1-propenyl, 1-i-propyl-2-propenyl, 1-methyl-2-c-pentenyl,1-methyl-3-c-pentenyl, 2-methyl-1-c-pentenyl, 2-methyl-2-c-pentenyl,2-methyl-3-c-pentenyl, 2-methyl-4-c-pentenyl, 2-methyl-5-c-pentenyl,2-methylene-c-pentyl, 3-methyl-1-c-pentenyl, 3-methyl-2-c-pentenyl,3-methyl-3-c-pentenyl, 3-methyl-4-c-pentenyl, 3-methyl-5-c-pentenyl,3-methylene-c-pentyl, 1-c-hexenyl, 2-c-hexenyl, 3-c-hexenyl or the likemay be mentioned.

A C₁₋₆ alkoxy group may be linear, branched or cyclic (a C₃₋₆cycloalkoxy group), and methoxy, ethoxy, n-propoxy, i-propoxy,c-propoxy, n-butoxy, i-butoxy, s-butoxy, t-butoxy, c-butoxy,1-methyl-c-propoxy, 2-methyl-c-propoxy, n-pentyloxy, 1-methyl-n-butoxy,2-methyl-n-butoxy, 3-methyl-n-butoxy, 1,1-dimethyl-n-propoxy,1,2-dimethyl-n-propoxy, 2,2-dimethyl-n-propoxy, 1-ethyl-n-propoxy,c-pentyloxy, 1-methyl-c-butoxy, 2-methyl-c-butoxy, 3-methyl-c-butoxy,1,2-dimethyl-c-propoxy, 2,3-dimethyl-c-propoxy, 1-ethyl-c-propoxy,2-ethyl-c-propoxy, n-hexyloxy, 1-methyl-n-pentyloxy,2-methyl-n-pentyloxy, 3-methyl-n-pentyloxy, 4-methyl-n-pentyloxy,1,1-dimethyl-n-butoxy, 1,2-dimethyl-n-butoxy, 1,3-dimethyl-n-butoxy,2,2-dimethyl-n-butoxy, 2,3-dimethyl-n-butoxy, 3,3-dimethyl-n-butoxy,1-ethyl-n-butoxy, 2-ethyl-n-butoxy, 1,1,2-trimethyl-n-propoxy,1,2,2-trimethyl-n-propoxy, 1-ethyl-1-methyl-n-propoxy,1-ethyl-2-methyl-n-propoxy, c-hexyloxy, 1-methyl-c-pentyloxy,2-methyl-c-pentyloxy, 3-methyl-c-pentyloxy, 1-ethyl-c-butoxy,2-ethyl-c-butoxy, 3-ethyl-c-butoxy, 1,2-dimethyl-c-butoxy,1,3-dimethyl-c-butoxy, 2,2-dimethyl-c-butoxy, 2,3-dimethyl-c-butoxy,2,4-dimethyl-c-butoxy, 3,3-dimethyl-c-butoxy, 1-n-propyl-c-propoxy,2-n-propyl-c-propoxy, 1-i-propyl-c-propoxy, 2-i-propyl-c-propoxy,1,2,2-trimethyl-c-propoxy, 1,2,3-trimethyl-c-propoxy,2,2,3-trimethyl-c-propoxy, 1-ethyl-2-methyl-c-propoxy,2-ethyl-1-methyl-c-propoxy, 2-ethyl-2-methyl-c-propoxy,2-ethyl-3-methyl-c-propoxy or the like may be mentioned.

A C₁₋₆ alkoxycarbonyl group may be linear, branched or cyclic (a C₃₋₆cycloalkoxycarbonyl group), and methoxycarbonyl, ethoxycarbonyl,n-propoxycarbonyl, i-propoxycarbonyl, c-propylcarbonyl,n-butoxycarbonyl, i-butoxycarbonyl, s-butoxycarbonyl, t-butoxycarbonyl,c-butoxycarbonyl, 1-methyl-c-propoxycarbonyl,2-methyl-c-propoxycarbonyl, n-pentyloxycarbonyl,1-methyl-n-butoxycarbonyl, 2-methyl-n-butoxycarbonyl,3-methyl-n-butoxycarbonyl, 1,1-dimethyl-n-propoxycarbonyl,1,2-dimethyl-n-propoxycarbonyl, 2,2-dimethyl-n-propoxycarbonyl,1-ethyl-n-propoxycarbonyl, c-pentyloxycarbonyl,1-methyl-c-butoxycarbonyl, 2-methyl-c-butoxycarbonyl,3-methyl-c-butoxycarbonyl, 1,2-dimethyl-c-propoxycarbonyl,2,3-dimethyl-c-propoxycarbonyl, 1-ethyl-c-propoxycarbonyl,2-ethyl-c-propoxycarbonyl, n-hexyloxycarbonyl,1-methyl-n-pentyloxycarbonyl, 2-methyl-n-pentyloxycarbonyl,3-methyl-n-pentyloxycarbonyl, 4-methyl-n-pentyloxycarbonyl,1,1-dimethyl-n-butoxycarbonyl, 1,2-dimethyl-n-butoxycarbonyl,1,3-dimethyl-n-butoxycarbonyl, 2,2-dimethyl-n-butoxycarbonyl,2,3-dimethyl-n-butoxycarbonyl, 3,3-dimethyl-n-butoxycarbonyl,1-ethyl-n-butoxycarbonyl, 2-ethyl-n-butoxycarbonyl,1,1,2-trimethyl-n-propoxycarbonyl, 1,2,2-trimethyl-n-propoxycarbonyl,1-ethyl-1-methyl-n-propoxycarbonyl, 1-ethyl-2-methyl-n-propoxycarbonyl,c-hexyloxycarbonyl, 1-methyl-c-pentyloxycarbonyl,2-methyl-c-pentyloxycarbonyl, 3-methyl-c-pentyloxycarbonyl,1-ethyl-c-butoxycarbonyl, 2-ethyl-c-butoxycarbonyl,3-ethyl-c-butoxycarbonyl, 1,2-dimethyl-c-butoxycarbonyl,1,3-dimethyl-c-butoxycarbonyl, 2,2-dimethyl-c-butoxycarbonyl,2,3-dimethyl-c-butoxycarbonyl, 2,4-dimethyl-c-butoxycarbonyl,3,3-dimethyl-c-butoxycarbonyl, 1-n-propyl-c-propoxycarbonyl,2-n-propyl-c-propoxycarbonyl, 1-i-propyl-c-propoxycarbonyl,2-i-propyl-c-propoxycarbonyl, 1,2,2-trimethyl-c-propoxycarbonyl,1,2,3-trimethyl-c-propoxycarbonyl, 2,2,3-trimethyl-c-propoxycarbonyl,1-ethyl-2-methyl-c-propoxycarbonyl, 2-ethyl-1-methyl-c-propoxycarbonyl,2-ethyl-2-methyl-c-propoxycarbonyl, 2-ethyl-3-methyl-c-propoxycarbonylor the like may be mentioned.

Specific preferred examples of the substituents R¹ and R³ which areindependent of each other are a hydrogen atom, a methyl group, a n-hexylgroup, a 2-fluoroethyl group, a benzyl group, a p-chlorophenyl group, ap-methylphenyl group, an ethoxycarbonylmethyl group, a 2-propenyl group,a 4-pyridylmethyl group, a 2-pyridylmethyl group, a methylcarbonylgroup, a benzoyl group, a 2-ethylaminoethyl group,2-(1-piperidino)-ethyl group and the like.

Specific preferred examples of the substituents R², R⁴ and R^(a) whichare independent of one another are a hydrogen atom, a hydroxyl group anda methoxy group.

Favorable compounds as the thrombopoietin receptor activator representedby the formula (1), the compound to be used in the preventive,therapeutic or improving agent for diseases against which activation ofthe thrombopoietin receptor is effective and the compound to be used inthe platelet increasing agent of the present invention are as follows.

1) Compounds represented by the formula (1) wherein each of each of R¹and R³ is independently a hydrogen atom, SO₃H, a C₁₋₆ alkyl group, aC₁₋₆ alkylcarbonyl group or a C₆₋₁₈ arylcarbonyl group (the C₁₋₆ alkylgroup, the C₁₋₆ alkylcarbonyl group and the C₆₋₁₈ arylcarbonyl group maybe optionally substituted with a hydroxyl group).

2) Compounds represented by the formula (1) wherein each of R¹ and R³ isindependently a hydrogen atom, SO₃H, a C₁₋₆ alkyl group, a C₁₋₆alkylcarbonyl group or a C₆₋₁₈ arylcarbonyl group (the C₁₋₆ alkyl group,the C₁₋₆ alkylcarbonyl group and the C₆₋₁₈ arylcarbonyl group may beoptionally substituted with NR⁹R¹⁰ (wherein each of R⁹ and R¹⁰ isindependently a hydrogen atom or a C₁₋₆ alkyl group (the C₁₋₆ alkylgroup may be optionally substituted with a C₆₋₁₈ aryl group) or R⁹ andR¹⁰ mean, together with each other, —(CH₂)_(n)X(CH₂)_(m)— (wherein X isCR¹¹R¹² (wherein each of R¹¹ and R¹² is independently a hydrogen atom ora C₁₋₆ alkyl group (the C₁₋₆ alkyl group may be optionally substitutedwith a C₆₋₁₈ aryl group)), NR¹³ (wherein R¹³ is a hydrogen atom or aC₁₋₆ alkyl group (the C₁₋₆ alkyl group may be optionally substitutedwith a C₆₋₁₈ aryl group)), O or S, n is 1, 2 or 3, and m is 1, 2 or 3,provided that n+m is 3, 4 or 5))).

3) Compounds represented by the formula (1) wherein each of R¹ and R³ isindependently a hydrogen atom or a C₁₋₆ alkyl group.

4) The compounds according to 3) wherein each of R¹ and R³ isindependently a hydrogen atom or a methyl group, and each of R² and R⁴is independently a hydrogen atom, a hydroxyl group or a methoxy group.

5) Compounds represented by the formula (1) or the compounds accordingto 1), 2), 3) or 4) wherein R² is a hydrogen atom.

6) Compounds according to 5) wherein each of R⁴ and R^(a) isindependently a hydrogen atom or a methoxy group.

7) Compounds wherein R¹, R², R³, R⁴ and R^(a) are any of the followingcombinations.

-   -   R¹═H, R²═OH, R³═H, R⁴═OH, R^(a)═H    -   R¹═CH₃, R²═OCH₃, R³═CH₃, R⁴═OCH₃, R^(a)═H    -   R¹═H, R²═OCH₃, R³═H, R⁴═OCH₃, R^(a)═H    -   R¹═CH₃, R²═OH, R³═CH₃, R⁴═OH, R^(a)═H    -   R¹═H, R²═H, R³═H, R⁴═OH, R^(a)═H    -   R¹═CH₃, R²═H, R³═CH₃, R⁴═OCH₃, R^(a)═H    -   R¹═CH₃, R²═H, R³═CH₃, R⁴═OH, R^(a)═H    -   R¹═H, R²═H, R³═H, R⁴═H, R^(a)═H    -   R¹═CH₃, R²═H, R³═CH₃, R⁴═H, R¹═H    -   R¹═H, R²═H, R³═CH₃, R⁴═H, R^(a)═H    -   R¹═CH₃, R²═H, R³═SO₃H, R⁴═H, R^(a)═H    -   R¹═CH₃, R²═H, R³═CH₃, R⁴═OCH₃, R^(a)═OCH₃

8) Compounds wherein R¹, R², R³, R⁴ and R^(a) are any of the followingcombinations.

-   -   R¹═H, R²═H, R³═H, R⁴═H, R^(a)═H    -   R¹═CH₃, R²═H, R³═CH₃, R⁴═H, R^(a)═H    -   R¹═H, R²═H, R³═CH₃, R⁴═H, R^(a)═H    -   R¹═CH₃, R²═H, R³═SO₃H, R⁴═H, R^(a)═H    -   R¹═CH₃, R²═H, R³═CH₃, R⁴═OCH₃, R^(a)═H    -   R¹═CH₃, R²═H, R³═CH₃, R⁴═OCH₃, R^(a)═OCH₃

9) Compounds represented by the formula (1) wherein both R² and R⁴ areboth hydroxyl groups, or R² is a hydroxyl group and R⁴ is hydrogen atom,and R¹ and R³ are both hydrogen atoms.

10) Compounds represented by the formula (1) wherein R² are R⁴ are bothC₁₋₆ alkoxy groups, or R² is a C₁₋₆ alkoxy group and R⁴ is hydrogenatom, and R¹ and R³ are both C₁₋₆ alkyl groups.

11) Compounds represented by the formula (1) wherein R² and R⁴ are bothhydrogen atoms, and R¹ and R³ are both hydrogen atoms, SO₃H, C₁₋₆ alkylgroups, C₁₋₆ alkylcarbonyl groups or C₆₋₁₈ arylcarbonyl groups (the C₁₋₆alkyl groups, the C₁₋₆ alkylcarbonyl groups and the C₆₋₁₈ arylcarbonylgroups may be optionally substituted with a halogen atom, a hydroxylgroup, a C₂₋₆ alkenyl group, a C₁₋₆ alkoxy group, a C₁₋₆ alkoxycarbonylgroup, a C₆₋₁₈ aryl group, a 2-pyridyl group, a 3-pyridyl group, a4-pyridyl group, a 2-furanyl group, a 3-furanyl group, a 2-thienylgroup, a 3-thienyl group (the C₆₋₁₈ aryl group, the 2-pyridyl group, the3-pyridyl group, the 4-pyridyl group, the 2-furanyl group, the 3-furanylgroup, the 2-thienyl group and the 3-thienyl group may be optionallysubstituted with a halogen atom or a C₁₋₆ alkyl group) or NR⁹R¹⁰(wherein each of R⁹ and R¹⁰ is independently a hydrogen atom or a C₁₋₆alkyl group (the C₁₋₆ alkyl group may be optionally substituted with aC₆₋₁₈ aryl group) or R⁹ and R¹⁰ mean, together with each other,—(CH₂)_(n)X(CH₂)_(m)— (wherein X is CR¹¹R¹² (wherein each of R¹¹ and R¹²is independently a hydrogen atom or a C₁₋₆ alkyl group (the C₁₋₆ alkylgroup may be optionally substituted with a C₆₋₁₈ aryl group)), NR¹³(wherein R¹³ is a hydrogen atom or a C₁₋₆ alkyl group (the C₁₋₆ alkylgroup may be optionally substituted with a C₆₋₁₈ aryl group)), O or S, nis 1, 2 or 3, and m is 1, 2 or 3, provided that n+m is 3, 4 or 5))).

12) Compounds represented by the formula (1) wherein R² and R⁴ are bothhydrogen atoms, R¹ is a methyl group, and R³ is a hydrogen atom, SO₃H, aC₁₋₆ alkyl group, a C₁₋₆ alkylcarbonyl group or a C₆₋₁₈ arylcarbonylgroup (the C₁₋₆ alkyl group, the C₁₋₆ alkylcarbonyl group and the C₆₋₁₈arylcarbonyl group may be optionally substituted with a halogen atom, ahydroxyl group, a C₂₋₆ alkenyl group, a C₁₋₆ alkoxy group, a C₁₋₆alkoxycarbonyl group, a C₆₋₁₈ aryl group, a 2-pyridyl group, a 3-pyridylgroup, a 4-pyridyl group, a 2-furanyl group, a 3-furanyl group, a2-thienyl group, a 3-thienyl group (the C₆₋₁₈ aryl group, the 2-pyridylgroup, the 3-pyridyl group, the 4-pyridyl group, the 2-furanyl group,the 3-furanyl group, the 2-thienyl group and the 3-thienyl group may beoptionally substituted with a halogen atom or a C₁₋₆ alkyl group) orNR⁹R¹⁰ (wherein each of R⁹ and R¹⁰ is independently a hydrogen atom or aC₁₋₆ alkyl group (the C₁₋₆ alkyl group may be optionally substitutedwith a C₆₋₁₈ aryl group) or R⁹ and R¹⁰ mean, together with each other,—(CH₂)_(n)X(CH₂)_(m)— (wherein X is CR¹¹R¹² (wherein each of R¹¹ and R¹²is independently a hydrogen atom or a C₁₋₆ alkyl group (the C₁₋₆ alkylgroup may be optionally substituted with a C₆₋₁₈ aryl group)), NR¹³(wherein R¹³ is a hydrogen atom or a C₁₋₆ alkyl group (the C₁₋₆ alkylgroup may be optionally substituted with a C₆₋₁₈ aryl group)), O or S, nis 1, 2 or 3, and m is 1, 2 or 3, provided that n+m is 3, 4 or 5))).

Favorable compounds represented by the formula (2) are as follows.

13) Compounds wherein R⁵, R⁶, R⁷, R⁸ and R^(b) are any of the followingcombinations.

-   -   R⁵═H, R⁶═OH, R⁷═H, R⁸═OH, R^(b)═H    -   R⁵═CH₃, R⁶═OCH₃, R⁷═CH₃, R⁸═OCH₃, R^(b)═H    -   R⁵═H, R⁶═OCH₃, R⁷═H, R⁸═OCH₃, R^(b)═H    -   R⁵═CH₃, R⁶═OH, R⁷═CH₃, R⁸═OH, R^(b)═H    -   R⁵═H, R⁶═H, R⁷═H, R⁸═OH, R^(b)═H    -   R⁵═CH₃, R⁶═H, R⁷═CH₃, R⁸═OCH₃, R^(b)═H    -   R⁵═CH₃, R⁶═H, R⁷═CH₃, R⁸═OH, R^(b)═H    -   R⁵═H, R⁶═H, R⁷═H, R⁸═H, R^(b)═H    -   R⁵═CH₃, R⁶═H, R⁷═CH₃, R⁸═H, R^(b)═H    -   R⁵═H, R⁶═H, R⁷═CH₃, R⁸═H, R^(b)═H    -   R⁵═CH₃, R⁶═H, R⁷═CH₃, R⁸═OCH₃, R^(b)═OCH₃

14) Compounds wherein R⁵, R⁶, R⁷, R⁸ and R^(b) are any of the followingcombinations.

-   -   R⁵═H, R⁶═H, R⁷═H, R⁸═H, R^(b)═H    -   R⁵═CH₃, R⁶═H, R⁷═CH₃, R⁸═H, R^(b)═H    -   R⁵═H, R⁶═H, R⁷═CH₃, R⁸═H, R^(b)═H    -   R⁵═CH₃, R⁶═H, R⁷═CH₃, R⁸═OCH₃, R^(b)═H    -   R⁵═CH₃, R⁶═H, R⁷═CH₃, R⁸═OCH₃, R^(b)═OCH₃

15) Compounds wherein R⁵, R⁶, R⁷, R⁸ and R^(b) are any of the followingcombinations.

-   -   R⁵═CH₃, R⁶═H, R⁷═CH₃, R⁸═H, R^(b)═H    -   R⁵═H, R⁶═H, R⁷═CH₃, R⁸═H, R^(b)═H    -   R⁵═CH₃, R⁶═H, R⁷═CH₃, R⁸═OCH₃, R^(b)═H    -   R⁵═CH₃, R⁶═H, R⁷═CH₃, R⁸═OCH₃, R^(b)═OCH₃

The compounds of the present invention represented by the formula (1) orpharmaceutically acceptable salts thereof may be in the form ofarbitrary crystals or arbitrary hydrates, depending on the productionconditions. The present invention covers these crystals, hydrates andmixtures. They may be in the form of solvates with organic solvents suchas acetone, ethanol and tetrahydrofuran, and the present inventioncovers any of these forms.

The compounds of the present invention represented by the formula (1)may be converted to pharmaceutically acceptable salts or may beliberated from the resulting salts, if necessary. The pharmaceuticallyacceptable salts of the present invention may be, for example, saltswith alkali metals (such as lithium, sodium and potassium), alkalineearth metals (such as magnesium and calcium), ammonium, organic basesand amino acids. They may be salts with inorganic acids (such ashydrochloric acid, hydrobromic acid, phosphoric acid and sulfuric acid)and organic acids (such as acetic acid, citric acid, maleic acid,fumaric acid, benzenesulfonic acid and p-toluenesulfonic acid).

The compounds which serve as prodrugs are derivatives of the presentinvention having chemically or metabolically degradable groups whichgive pharmacologically active compounds of the present invention uponsolvolysis or under physiological conditions in vivo. Methods forselecting or producing appropriate prodrugs are disclosed, for example,in Design of Prodrug (Elsevier, Amsterdam 1985). In the presentinvention, when the compound has a hydroxyl group, acyloxy derivativesobtained by reacting the compound with appropriate acyl halides orappropriate acid anhydrides may, for example, be mentioned as prodrugs.Acyloxys particularly preferred as prodrugs include —OCOC₂H₅,—OCO(t-Bu), —OCOC₁₅H₃₁, —OCO(m-CO₂Na-Ph), —OCOCH₂CH₂CO₂Na,—OCOCH(NH₂)CH₃, —OCOCH₂N(CH₃)₂ and the like. When the compound of thepresent invention has an amino group, amide derivatives obtained byreacting the compound having an amino group with appropriate acidhalides or appropriate mixed acid anhydrides may, for example, bementioned as prodrugs. Amides particularly preferred as prodrugs include—NHCO(CH₂)₂₀OCH₃, —NHCOCH(NH₂)CH₃ and the like.

Medicines containing the thrombopoietin receptor activators of thepresent invention or prodrugs, pharmaceutically acceptable salts orsolvates thereof as an active ingredient may usually be administered asoral medicines such as tablets, capsules, powder, granules, pills andsyrup, as rectal medicines, percutaneous medicines or injections. Theagents of the present invention may be administered as a singletherapeutic agent or as a mixture with other therapeutic agents. Thoughthey may be administered as they are, they are usually administered inthe form of medical compositions. These pharmaceutical preparations canbe obtained by adding pharmacologically and pharmaceutically acceptableadditives by conventional methods. Namely, for oral medicines, ordinaryexcipients, lubricants, binders, disintegrants, humectants, plasticizersand coating agents may be used. Oral liquid preparations may be in theform of aqueous or oily suspensions, solutions, emulsions, syrups orelixirs or may be supplied as dry syrups to be mixed with water or otherappropriate solvents before use. Such liquid preparations may containordinary additives such as suspending agents, perfumes, diluents andemulsifiers. In the case of rectal administration, they may beadministered as suppositories. Suppositories may use an appropriatesubstance such as cacao butter, laurin tallow, Macrogol, glycerogelatin,Witepsol, sodium stearate and mixtures thereof as the base and may, ifnecessary, contain an emulsifier, a suspending agent, a preservative andthe like. For injections, pharmaceutical ingredients such as distilledwater for injection, physiological saline, 5% glucose solution,propylene glycol and other solvents or solubilizing agents, a pHregulator, an isotonizing agent and a stabilizer may be used to formaqueous dosage forms or dosage forms which need dissolution before use.

The dose of the agents of the present invention for administration tohuman is usually about from 0.1 to 1000 mg/human/day in the case of oralmedicines or rectal administration and about from 0.05 mg to 500mg/human/day in the case of injections, though it depends on the age andconditions of the patient. The above-mentioned ranges are mere examples,and the dose should be determined from the conditions of the patient.

The present invention is used when the use of compounds which havethrombopoietin receptor affinity and act as thrombopoietin receptoragonists are expected to improve pathological conditions. For example,hematological disorders accompanied by abnormal platelet count may bementioned. Specifically, it is effective for therapy or prevention ofhuman and mammalian diseases caused by abnormal megakaryopoiesis,especially those accompanied by thrombocytopenia. Examples of suchdiseases include thrombocytopenia accompanying chemotherapy orradiotherapy of cancer, thrombocytopenia caused by bone marrowtransplantation, surgery and serious infections, or gastrointestinalbleeding, but such diseases are not restricted to those mentioned.Typical thrombocytopenias such as aplastic anemia, idiopathicthrombocytopenic purpura, myelodysplastic syndrome and thrombopoietindeficiency are also targets of the agents of the present invention. Thepresent invention may be used as a peripheral stem cell mobilizer, amegakaryoblastic or megakaryocytic leukemia cell differentiation inducerand a platelet increasing agent for platelet donors. In addition,potential applications include therapeutic angiogenesis based ondifferentiation and proliferation of vascular endothelial cells andendothelial progenitor cells, prevention and therapy ofarteriosclerosis, myocardial infarction, unstable angina, peripheralartery occlusive disease, but there is no restriction.

Next, the process for producing a compound represented by the formula(2) will be described.

The compound represented by the formula (2) is produced by incubating amicroorganism belonging to the Basipetospora genus and isolating thecompound from the culture medium.

The microorganism belonging to the Basipetospora genus to be used in thepresent invention may be a variant such as a strain with higherproductivity obtained by inducing mutation in a microorganism or astrain obtained by transformation of a heterologous or homologous hostusing an appropriate vector such as a plasmid carrying a gene associatedwith the reaction which produces the compound from cells of a strain.

Preferred microorganisms belonging to the Basipetospora genus arestrains of Basipetospora sp., and Basipetospora sp. strain No. 1142 isparticularly preferred.

Bacteriological characteristics of Basipetospora sp. strain No. 1142 areas follows.

-   1. Viability in various media-   Viability at 25° C. after 7 days of incubation

Oatmeal agar medium + Malt extract agar medium + Potato dextrose agarmedium + Viability at 37° C. − Production and secretion of soluble dye −

-   Colony morphology: diameter 37-42 cm, white and wooly, smooth, white    bottoms-   2. Morphological characteristics under a microscope Hyphae: 1-5 μm    wide, colorless, smooth and branched, septate-   Conidiophores: upright, monogenetic-   Conidiogenous cells: linear, nonannular ends-   Conidia: connected in chains, unicellular, spherical to    subspherical, rough surface, from 7 to 12 μm in diameter

On the basis of the above-mentioned characteristics, Strain No. 1142 wasclassified into the genus Basipetospora, in comparison with knownstrains, and designated as Basipetospora sp. strain No. 1142. It wasdeposited as follows.

-   The International Patent Organism Depositary (IPOD), the National    Institute of Advanced Industrial Science and Technology,-   Chuo 6, 1-1-1, Higashi, Tsukuba-shi, Ibaraki, Japan, 305-8566-   Jul. 22, 2002 (22.07.2002), 14 Sanseiki, No. 990 (FERM P-18940)-   Accession Number: IPOD FERM BP-8410

Basipetospora sp. strain No. 1142 is likely to change itscharacteristics, like other mold fungi. For example, any mutants(spontaneous or induced), transformants or genetic recombinants of ororiginated from strain No. 1142 may be used in the present invention aslong as they produce xanthocillin X monomethyl ether (the compoundwherein R⁵═H, R⁶═H, R⁷═CH₃, R⁸═H and R^(b)═H), xanthocillin X dimethylether (the compound wherein R⁵═CH₃, R⁶═H, R⁷═CH₃, R⁸═H and R^(b)═H),3-methoxyxanthocillin X dimethyl ether (the compound wherein R⁵═CH₃,R⁶═H, R⁷═CH₃, R⁸═OCH₃ and R^(b)═H) or 3,3′-dimethoxyxanthocillin Xdimethyl ether (the compound wherein R⁵═CH₃, R⁶═H, R⁷═CH₃, R⁸═OCH₃ andR^(b)═OCH₃).

The method of incubating a microorganism of Basipetospora sp. will bedescribed.

In the present invention, to obtain the compound represented by theformula (2), a microorganism of Basipetospora sp. is incubated in aculture medium. Known nutrients conventionally used for incubation ofmold fungi may be used. For example, the carbon source may, for example,be glucose, sucrose, starch syrup, starch, dextrin, soybean oil, and thenitrogen source may, for example, be peptone, broth, yeast extract, cornsteep liquor, oatmeal, ammonium sulfate, ammonium chloride, ammoniumnitrate or urea. If necessary, inorganic salts such as sodium chloride,magnesium sulfate, copper sulfate, zinc sulfate, manganese chloride,calcium carbonate and phosphate salts may be added singly or incombination. In addition, organic substances which promote growth of thestrain or production of the compound represented by the formula (2) suchas nucleic acids, vitamins and inorganic substances may be arbitrarilyadded. When foaming of the culture remarkable, an antifoam agent may beadded.

As the incubation method, incubation with shaking or with stirring andaeration under aerobic conditions is most suitable. The incubationtemperature is preferably from 20 to 28° C., and the culture medium ispreferably adjusted to pH 5.0-8.0. As to the incubation time, it ispreferred to continue the incubation usually for 8 to 16 days until theaccumulation of the compounds represented by the formula (2) in theculture medium or the cells reaches a maximum. Suitable incubationconditions may be selected depending on the characteristics of thestrain to be used and the incubation method.

The methods of isolation and purification of the compounds representedby the formula (2) will be described. After the incubation, thecompounds represented by the formula (2) is isolated and purified fromthe culture medium by ordinary methods for isolation of microbialmetabolites from the culture. Namely, vacuum concentration, freezedrying, extraction with organic solvents such as butanol, ethyl acetate,chloroform and benzene, various kinds of ion exchange chromatography,gel permeation chromatography using Sephadex LH-20 and the like,adsorption chromatography using activated carbon, silica gel and thelike, absorption and desorption of active substances by thin layerchromatography and high performance liquid chromatography usingreversed-phase columns may be employed singly or in appropriatecombinations or repeatedly to isolate the compounds represented by theformula (2). The physical and chemical properties of some of thecompounds represented by the formula (2) are shown below.

-   Xanthocillin X monomethyl ether (the compound wherein R⁵═H, R⁶═H,    R⁷═CH₃, R⁸═H and R^(b)═H)-   1. Molecular weight: 302-   2. Composition formula: C₁₉H₁₄N₂O₂-   3. Properties and color: yellow neutral substance in the form of    needle crystals-   4. ¹H Nuclear magnetic resonance spectrum: The chemical shifts (δ)    measured by proton nuclear magnetic resonance spectroscopy in    deuteriochloroform are shown below.-   δ (ppm): 7.79 (2H, d), 7.72 (2H, d), 7.02 (2H, d), 7.01 (1H, s),    6.87 (1H, s), 6.86 (2H, d), 3.85 (3H, s)-   5. ¹³C Nuclear magnetic resonance spectrum: The chemical shifts (δ)    measured by carbon nuclear magnetic resonance spectroscopy in    deuteriochloroform are shown below.-   δ (ppm): 48.0, 114.1, 115.2, 115.6, 116.1, 123.6, 126.9, 127.7,    131.4, 131.7, 159.8, 161.4, 173.4, 173.5-   6. Mass spectrum: (EI-MS)m/z=302 (M⁺) Xanthocillin X dimethyl ether    (the compound wherein R⁵═CH₃, R⁶═H, R⁷═CH₃, R⁸═H and R^(b)═H)-   1. Molecular weight: 316-   2. Composition formula: C₂₀H₁₆N₂O₂-   3. Properties and color: yellow neutral substance in the form of    needle crystals-   4. ¹H Nuclear magnetic resonance spectrum: The chemical shifts (δ)    measured by proton nuclear magnetic resonance spectroscopy in    deuteriochloroform are shown below.-   δ (ppm): 7.79 (4H, d), 7.02 (2H, d), 6.98 (4H, d), 3.87 (6H, s)-   5. ¹³C Nuclear magnetic resonance spectrum: The chemical shifts (δ)    measured by carbon nuclear magnetic resonance spectroscopy in    deuteriochloroform are shown below.-   δ (ppm): 55.5, 114.5, 116.3, 124.9, 127.5, 131.8, 161.2, 173.3-   6. Mass spectrum: (EI-MS)m/z=316 (M⁺) 3-Methoxyxanthocillin X    dimethyl ether (the compound wherein R⁵═CH₃, R⁶═H, R⁷═CH₃, R⁸OCH₃    and R^(b)═H)-   1. Molecular weight: 346-   2. Composition formula: C₂₁H₁₈N₂O₃-   3. Properties and color: brown neutral substance-   4. ¹H Nuclear magnetic resonance spectrum: The chemical shifts (δ)    measured by proton nuclear magnetic resonance spectroscopy in    deuteriochloroform are shown below.-   δ (ppm): 7.79 (2H, s), 7.49 (H, d), 7.36 (H, dd), 7.03 (H, s), 7.02    (H, s), 6.99 (2H, d), 6.95 (H, d), 3.96 (3H, s), 3.95 (3H, s), 3.88    (3H, s)-   5. ¹³C Nuclear magnetic resonance spectrum: The chemical shifts (δ)    measured by carbon nuclear magnetic resonance spectroscopy in    deuteriochloroform are shown below.-   δ (ppm): 55.5, 56.1, 111.2, 111.9, 114.5, 116.3, 116.2, 124.6,    124.9, 125.1, 127.6, 127.8, 131.8, 149.1, 150.9, 161.2, 173.3, 173.5-   6. Mass spectrum: (EI-MS)m/z=346 (M⁺) 3,3′-Dimethoxyxanthocillin X    dimethyl ether (the compound wherein R⁵═CH₃, R⁶═H, R⁷═CH₃, R⁸═OCH₃    and R^(b)═OCH₃)-   1. Molecular weight: 376-   2. Composition formula: C₂₂H₂₀N₂O₄-   3. Properties and color: brown neutral substance-   4. ¹H Nuclear magnetic resonance spectrum: The chemical shifts (δ)    measured by proton nuclear magnetic resonance spectroscopy in    deuteriochloroform are shown below.-   δ (ppm): 7.79 (2H, d), 7.08 (2H, s), 7.05 (H, s), 7.00 (H, s), 6.99    (2H, d), 3.92 (9H, s), 3.87 (3H, s)-   5. ¹³C Nuclear magnetic resonance spectrum: The chemical shifts (δ)    measured by carbon nuclear magnetic resonance spectroscopy in    deuteriochloroform are shown below.-   δ (ppm): 55.5, 56.2, 61.1, 107.4, 114.6, 116.0, 117.5, 124.7, 127.5,    127.8, 128.3, 132.0, 140.1, 153.3, 161.4, 173.5, 173.9-   6. Mass spectrum: (EI-MS)m/z=376 (M⁺)

Now, production of the compounds represented by the formula (1) will bedescribed.

The compounds represented by the formula (1) are obtainable usingmicroorganisms, by chemical modification of microbially producedcompounds, or by chemical synthesis.

The compound wherein R¹═H, R²═H, R³═H, R⁴═H and R^(a)═H (xanthocillin X)is obtainable using a microorganism such as Penicillium notatum orPenicillium Chrysogenum (GB 898498, Antibiotics Ann (1957) Volume Date1956-57 140-3p and DE 1123087).

The compound wherein R¹═H, R²═H, R³═CH₃, R⁴═H and R^(a)═H (xanthocillinX monomethyl ether) is obtainable using a microorganisms such asDichotomomyces albus (J. Antibiotics 21, 582-587 (1968), J. Antibiotics21 587-591 (1968)).

The compound wherein R¹═CH₃, R²═H, R³═CH₃, R⁴═H and R^(a)═H(xanthocillin X dimethyl ether), the compound wherein R¹═C₂H₅, R²═H,R³═C₂H₅, R⁴═H and R^(a)═H (xanthocillin X diethyl ether) and thecompound wherein R¹═CH₃, R²═H, R³═CH₃, R⁴═OCH₃ and R^(a)═H(3-methoxyxanthocillin X dimethyl ether) are obtainable using amicroorganisms such as Aspergullus sp. (Strain No. 208 or No. 98) (J.Antibiotics 21 (12) 671-679 (1968)).

The compound wherein R¹═H, R²═OH, R³═H, R⁴═H and R^(a)═H (xanthocillinY1) and the compound wherein R¹═H, R²═OH, R³═H, R⁴═OH and R^(a)═H(xanthocillin Y2) are obtainable using a microorganism such asPenicillium notatum (Chem. Ber 105 (9), 3061 (1972)).

The compound wherein R¹═CH₃, R²═H, R³═SO₃H, R⁴═H and R^(a)═H(xanthocillin X monomethyl ether sulfate ester) is obtainable using amicroorganism such as Aspergillus sp. (No. FA2692) (J. Antibiotics 46687-688 (1993)).

Among the compounds represented by the formula (1), the followingcompounds are obtained by chemical modification of microbially producedcompounds.

The compounds wherein R¹═CH₃, R²═H, R⁴═H, R^(a)═H and R³ is an alkylgroup, an alkylcarbonyl group or an arylcarbonyl group are obtainablefrom the xanthocillin X monomethyl ether by the method disclosed inJP-A-2-304058.

Namely, they are obtainable by reacting the corresponding halide withxanthocillin X monomethyl ether in the presence of a base such asK₂CO_(3.)

The compounds wherein R²═H, R⁴═H, R^(a)═H, and R¹ and R³ arealkylcarbonyl groups or arylcarbonyl groups are obtainable fromxanthocillin X by the method disclosed in Pharmazie 12, 567-580 (1957).

Namely, they are obtainable by reacting the corresponding acid anhydrideor acyl halide with xanthocillin X in the presence of an appropriatebase.

The compounds wherein R²═H, R⁴═H, R^(a)═H, and R¹ and R³ are alkylgroups substituted by dialkylamino groups are obtainable fromxanthocillin X by the method disclosed in DE 11658.

Namely, they are obtainable by reacting the corresponding halide withxanthocillin X.

The compounds wherein R²═H, R⁴═H, R^(a)═H, and R¹ and R³ are alkylgroups, the compounds wherein R² is an alkoxy group, R⁴═H, R^(a)═H, andR¹ and R³ are alkyl groups, and the compounds wherein R^(a)═H, R² and R⁴are alkoxy groups, and R¹ and R³ are alkyl groups are obtainable fromxanthocillin X, xanthocillin Y1 or xanthocillin Y2, respectively, by themethod disclosed in Chem Ber 105 (9) 3061 (1972).

Namely, they are obtainable by reacting the corresponding diazoalkylwith xanthocillin X, xanthocillin Y1 or xanthocillin Y2, respectively.

Among the compounds represented by the formula (1), the compound whereinR¹═CH₃, R²═H, R³═CH₃, R⁴═H and R^(a)═H (xanthocillin X dimethyl ether)is obtainable by chemical synthesis (Angew Chem 74, 215 (1962), Chem Ber98 (1) 193-201 (1965) and DE 1167332).

EXAMPLES

Now, the present invention will be described in further detail byreference to Examples. However, it should be understood that the presentinvention is by no means restricted to these specific Examples.

Preparation Example 1

100 ml of a liquid medium (composition; soluble starch 2%, glucose 0.5%,polypeptone 0.2%, corn steep liquor 0.5%) was poured into a 500 mlErlenmeyer flask and sterilized by autoclaving at 120° C. for 20minutes, then inoculated with a loopful of strain No. 1142 of theBasipetospora genus and incubated at 25° C. for 4 days with rotation at140 rpm. The resulting culture was transferred to 1 l of the sameculture medium as mentioned above in 5 l Erlenmeyer flasks (18 flaskssterilized by autoclaving) and incubated at 25° C. for 7 days withrotation at 140 rpm. After incubation, the resulting culture wasfiltered to give cells and a filtrate.

The cells were extracted with 5 l of 80% aqueous acetone, and the cellswere removed by filtration to obtain a cell extract. The cell extractwas concentrated by removing the solvent in vacuo, and 0.5 l of hexanewas added. The resulting aqueous layer was extracted with 0.5 l of ethylacetate three times, and the ethyl acetate layer was concentrated invacuo to give about 771 mg of a brown oil. The oil was separated bysilica gel chromatography (2.5×28 cm) using chloroform-methanol(100:0-5) as the developing solvent, and each fraction was concentratedto dryness. Thus, yellow active fraction A (about 33 mg) containingxanthocillin X monomethyl ether and yellow active fraction B (about 37mg) containing xanthocillin X dimethyl ether were obtained.

Crystallization of active fraction B from chloroform-hexane gave yellowneedle crystals of xanthocillin X dimethyl ether (about 30 mg). Activefraction A was separated by silica gel chromatography (2.5×28 cm) usingchloroform-methanol (3:1-3) as the developing solvent, and each fractionwas concentrated to dryness. Thus, active fraction (about 11 mg)containing xanthocillin X monomethyl ether was obtained. Crystallizationof this fraction from chloroform-hexane gave yellow needle crystals ofxanthocillin X monomethyl ether (about 10 mg).

The physical and chemical properties of the compounds thus obtained areshown below.

-   1) The physical and chemical properties of the xanthocillin X    monomethyl ether:-   1. Molecular weight: 302-   2. Composition formula: C₁₉H₁₄N₂O₂-   3. Properties and color: yellow neutral substance in the form of    needle crystals-   4. ¹H Nuclear magnetic resonance spectrum: The chemical shifts (δ)    measured by proton nuclear magnetic resonance spectroscopy in    deuteriochloroform are shown below.-   δ (ppm): 7.79 (2H, d), 7.72 (2H, d), 7.02 (2H, d), 7.01 (1H, s),    6.87 (1H, s), 6.86 (2H, d), 3.85 (3H, s)-   5. ¹³C Nuclear magnetic resonance spectrum: The chemical shifts (δ)    measured by carbon nuclear magnetic resonance spectroscopy in    deuteriochloroform are shown below.-   δ (ppm): 48.0, 114.1, 115.2, 115.6, 116.1, 123.6, 126.9, 127.7,    131.4, 131.7, 159.8, 161.4, 173.4, 173.5-   6. Mass spectrum: (EI-MS)m/z=302 (M⁺)-   2) The physical and chemical properties of the xanthocillin X    dimethyl ether:-   1. Molecular weight: 316-   2. Composition formula: C₂₀H₁₆N₂O₂-   3. Properties and color: yellow neutral substance in the form of    needle crystals-   4. ¹H Nuclear magnetic resonance spectrum: The chemical shifts (δ)    measured by proton nuclear magnetic resonance spectroscopy in    deuteriochloroform are shown below.-   δ (ppm): 7.79 (4H, d), 7.02 (2H, d), 6.98 (4H, d), 3.87 (6H, s)-   5. ¹³C Nuclear magnetic resonance spectrum: The chemical shifts (δ)    measured by carbon nuclear magnetic resonance spectroscopy in    deuteriochloroform are shown below.-   δ (ppm): 55.5, 114.5, 116.3, 124.9, 127.5, 131.8, 161.2, 173.3-   6. Mass spectrum: (EI-MS)m/z=316 (M⁺)

Preparation Example 2

100 ml of a liquid medium (composition; soluble starch 2%, glucose 0.5%,yeast extract 0.2%, magnesium phosphate 1%, defatted soybean 1%) waspoured into a 500 ml Erlenmeyer flask and sterilized by autoclaving at120° C. for 20 minutes, then inoculated with a loopful of strain No.1142 of the Basipetospora genus and incubated at 25° C. for 4 days withrotation at 140 rpm. The resulting culture was transferred to 0.9 l ofthe same culture medium as mentioned above in 5 l Erlenmeyer flasks (5flasks sterilized by autoclaving) and incubated at 25° C. for 11 dayswith rotation at 140 rpm. After incubation, the resulting culture wasfiltered to give cells and a filtrate.

The cells were extracted with 1.5 l of 80% aqueous acetone, and thecells were removed by filtration to obtain a cell extract. The cellextract was concentrated by removing the solvent in vacuo, and 0.4 l ofhexane was added. The resulting aqueous layer was extracted with 0.8 lof ethyl acetate three times, and the ethyl acetate layer wasconcentrated in vacuo to give about 608 mg of a brown oil. The oil wasseparated by silica gel chromatography (2.5×28 cm) usingchloroform-methanol (100:0-5) as the developing solvent, and eachfraction was concentrated to dryness. Thus, a brown active fraction(about 34 mg) containing 3-methoxyxanthocillin X dimethyl ether and3,3′-dimethoxyxanthocillin X dimethyl ether was obtained.

This fraction was dissolved in acetonitrile and separated by C₁₈reversed phase preparative HPLC (eluent; 75 aqueous acetonitrile,elution rate; 15 ml/min, absorptiometric detector, column; InertsilPREP-ODS 20×250 mm, column temperature; 40° C.) to obtain a fractionwith a retention time of 13.3 to 14.0 minutes. The fraction with aretention time of 13.3 to 14.0 minutes was concentrated to dryness,dissolved in acetonitrile and purified by reversed phase preparativeHPLC (eluent; 65% aqueous acetonitrile, elution rate; 15 ml/min,absorptiometric detector, column; Inertsil PREP-ODS 20×250 mm, columntemperature; 40° C.) to obtain two fractions with retention times of25.4 minutes and 27.4 minutes. The fractions with retention times of25.4 minutes and 27.4 minutes were concentrated to dryness,respectively, to give 3-methoxyxanthocillin X dimethyl ether (about 3.6mg) as a brown substance and 3,3′-dimethoxyxanthocillin X dimethyl ether(about 5.5 mg) as a brown substance.

The physical and chemical properties of the compounds thus obtained areshown below.

-   1) The physical and chemical properties of the 3-methoxyxanthocillin    X dimethyl ether:-   1. Molecular weight: 346-   2. Composition formula: C₂₁H₁₈N₂O₃-   3. Properties and color: brown neutral substance-   4. ¹H Nuclear magnetic resonance spectrum: The chemical shifts (δ)    measured by proton nuclear magnetic resonance spectroscopy in    deuteriochloroform are shown below.-   δ (ppm): 7.79 (2H, s), 7.49 (H, d), 7.36 (H, dd), 7.03 (H, s), 7.02    (H, s), 6.99 (2H, d), 6.95 (H, d), 3.96 (3H, s), 3.95 (3H, s), 3.88    (3H, s)-   5. ¹³C Nuclear magnetic resonance spectrum: The chemical shifts (δ)    measured by carbon nuclear magnetic resonance spectroscopy in    deuteriochloroform are shown below.-   δ (ppm): 55.5, 56.1, 111.2, 111.9, 114.5, 116.3, 116.2, 124.6,    124.9, 125.1, 127.6, 127.8, 131.8, 149.1, 150.9, 161.2, 173.3, 173.5-   6. Mass spectrum: (EI-MS)m/z=346 (M⁺)-   2) The physical and chemical properties of the    3,3′-dimethoxyxanthocillin X dimethyl ether-   1. Molecular weight: 376-   2. Composition formula: C₂₂H₂₀N₂O₄-   3. Properties and color: brown neutral substance-   4. ¹H Nuclear magnetic resonance spectrum: The chemical shifts (δ)    measured by proton nuclear magnetic resonance spectroscopy in    deuteriochloroform are shown below.-   δ (ppm): 7.79 (2H, d), 7.08 (2H, s), 7.05 (H, s), 7.00 (H, s), 6.99    (2H, d), 3.92 (9H, s), 3.87 (3H, s)-   5. ¹³C Nuclear magnetic resonance spectrum: The chemical shifts (δ)    measured by carbon nuclear magnetic resonance spectroscopy in    deuteriochloroform are shown below.-   δ (ppm): 55.5, 56.2, 61.1, 107.4, 114.6, 116.0, 117.5, 124.7, 127.5,    127.8, 128.3, 132.0, 140.1, 153.3, 161.4, 173.5, 173.9-   6. Mass spectrum: (EI-MS)m/z=376 (M⁺)

Assay Example 1

Stimulation of Proliferation of a Thrombopoietin (TPO)-Dependent CellLine (1)

(1) Cells and Cell Culture

The reactivity of a compound of the present invention, xanthocillin Xmonomethyl ether, with thrombopoietin (TPO) receptor was assayed using ahuman leukemic cell line, UT7/EPO-mpl. UT7/EPO-mpl is a stabletransformed cell line obtained by introducing into human leukemic cellline UT7/EPO a vector that induces expression of human TPO receptor(c-mpl) under control of a cytomegaloviral promoter by the method ofTakatoku et al. (J. Biol. Chem., 272:7259-7263 (1997)). Proliferation ofthis cell line is stimulated by TPO, while its mother cell line UT7/EPOexhibits no response to TPO. These two cell lines were subcultured inIMDM (GIBCO) containing 10% fetal bovine serum (FBS; TRACE SCIENTIFIC)using a CO₂ incubator (5% CO₂, 37° C.).

(2) Cell Proliferation Assay by the MTT Method

The subcultured cells described above were washed twice with PBS andsuspended in IMDM containing 10% FBS at a cell density of 6×10⁴cells/ml. The cell suspension was transferred to a 96-well tissueculture plate (CORNING) in 100-μl aliquots. Then, xanthocillin Xmonomethyl ether dissolved in DMSO was diluted 83-fold with IMDMcontaining 10% FBS and added to the aforementioned cell suspension in20-μl aliquots. The suspension was incubated in a CO₂ incubator (5% CO₂,37° C.) for 4 days. Cell proliferation was assayed according to themethod of Mosmann et al. (J. Immunological Methods, 65:55-63 (1983)). A10-μl aliquot of 5 mg/ml MTT reagent (SIGMA) was added to each well ofthe tissue culture plate and the plate was incubated at 37° C. for 4 h.The formazan pigment generated was dissolved by adding 150 μl per wellof 0.1 M/L HCl/isopropanol solution and the absorbance of the resultingpigment solution was measured at 550 nm with a 96-well microplate reader(BIO-RAD, M450). FIG. 1 shows the results with UT7/EPO-mpl cells, whileFIG. 2 shows data obtained with UT7/EPO cells expressing no TPOreceptor.

Assay Example 2

Activity of Signal Transduction Mediated by TPO Receptor

The signal-transducing activity of a compound of the present invention,xanthocillin X monomethyl ether, mediated by TPO receptor was assayedaccording to the method of Komatsu et al. (Blood, 87:4552-4560 (1996)).Human leukemic cell line UT7/EPO-mpl was washed three times with PBS andsuspended in IMDM containing 10% FBS at a cell density of 9×10⁵cells/ml. The cell suspension was incubated in a CO₂ incubator (5% CO₂,37° C.) for 18 h. To 2 ml of this cell suspension (7×10⁶ cells/ml),either TPO (final concentration, 30 ng/ml) or a DMSO solution ofxanthocillin X monomethyl ether (final concentration, 1 μg/ml) wasadded. After incubating the mixture at 37° C. for 1-15 min, the cellswere lysed in 1.4 ml of TNE buffer (20 mM Tris-HCl buffer (pH 7.4)containing 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM PMSF, 1 mMNa₃VO₄, and 1/400-diluted Protease Inhibitor Cocktail (SIGMA)). The celllysate was centrifuged to collect the supernatant forimmunoprecipitation with antibodies against proteins involved in signaltransduction (anti-Jak2 (UPSTATE BIOTECHNOLOGY), anti-Tyk2 (UPSTATEBIOTECHNOLOGY), anti-STAT3 (SANTA CRUZ BIOTECHNOLOGY), anti-STAT5A(UPSTATE BIOTECHNOLOGY) and anti-PLCγ1 (UPSTATE BIOTECHNOLOGY)) andprotein G Sepharose (PHARMACIA). The immunoprecipitated protein fractionwas collected and denatured in a sample buffer for separation bySDS-polyacrylamide gel electrophoresis (7.5%). The separated proteinswere transferred onto PVDF membrane (ATTO, 0.2 μm) at 100 V for 1 h fordetection of tyrosine phosphorylation using an alkalinephosphatase-labeled antibody against phosphorylated tyrosine (RC20,TRANSDUCTION LABORATORIES). The antigen-antibody complex formed on thePVDF membrane was visualized with 150 μg/ml NBT (BIO-RAD) and 300 μg/mlBCIP (BIO-RAD). The results are summarized in Table 1.

TABLE 1 Xanthocillin X monomethyl DMSO ether TPO Jak2 − + + Tyk2 − + +STAT 3 − + + STAT 5A − + + PLCγ1 − + +

FIG. 1 demonstrated that proliferation of TPO-responsive UT7/EPO-mplcells was stimulated by xanthocillin X monomethyl ether in aconcentration-dependent manner, while no effect of this compound onproliferation was observed with UT7/EPO, the mother cell line, as shownin FIG. 2. These results indicate that the compound of the presentinvention, xanthocillin X monomethyl ether, acts on the TPO receptorselectively as an activator.

Table 1 shows that xanthocillin X monomethyl ether stimulatesphosphorylation of Jak2, Tyk2, STAT3, STAT5A and PLCγ1 in the samemanner as TPO does. The results demonstrate that the compound of thepresent invention shows agonistic action through the same signaltransduction as that caused by TPO.

Assay Example 3

Stimulation of Proliferation of a Thrombopoietin (TPO)-Dependent CellLine (2)

The reactivity of a compound of the present invention,3-methoxyxanthocillin X dimethyl ether, with thrombopoietin (TPO)receptor was assayed using a human leukemic cell line, UT7/EPO-mpl.

Cells subcultured in the same manner as in Assay Example 1(1) werewashed twice with PBS and suspended in IMDM containing 10% FBS at a celldensity of 6×10⁴ cells/ml. The cell suspension was transferred to a96-well tissue culture plate (CORNING) in 100-μl aliquots. Then,3-methoxyxanthocillin X dimethyl ether dissolved in DMSO was diluted83-fold with IMDM containing 10% FBS and added to the aforementionedcell suspension in 20-μl aliquots. The suspension was incubated in a CO₂incubator (5% CO₂, 37° C.) for 4 days. Cell proliferation was assayedusing WST-8 reagent (Kishida Chemical Co., Ltd.) according toinstructions by the manufacturer. A 10-μl aliquot of 5 mM WST-8 reagentsolution was added to each well of the tissue culture plate, and theplate was incubated at 37° C. for 4 h. The formazan pigment generatedwas detected by measuring the absorbance at 450 nm with a 96-wellmicroplate reader (Nihon Molecular Devices, Spectramax 190). FIG. 3shows the results with UT7/EPO-mpl cells, while FIG. 4 shows dataobtained with UT7/EPO cells expressing no TPO receptor.

FIG. 3 demonstrated that proliferation of TPO-responsive UT7/EPO-mplcells was stimulated by 3-methoxyxanthocillin X dimethyl ether in aconcentration-dependent manner, while no effect of this compound onproliferation was observed with UT7/EPO, the mother cell line, as shownin FIG. 4. These results indicate that the compound of the presentinvention, 3-methoxyxanthocillin X dimethyl ether, acts on the TPOreceptor selectively as an activator.

Formulation Example 1

A granule preparation containing the following ingredients is prepared.

Ingredients Compound represented by the formula (1)  10 mg Lactose  700mg Corn Starch  274 mg HPC-L  16 mg 1000 mg

A compound represented by the formula (1) and lactose are sifted througha 60-mesh sieve. Corn starch is sifted though a 120-mesh sieve. They aremixed in a V-type blender. The powder mixture is kneaded with alow-viscosity hydroxypropylcellulose (HPC-L) aqueous solution,granulated (extrusion granulation, die size 0.5-1 mm) and dried. Theresulting dry granules are sifted through a shaking sieve (12/60 mesh)to obtain a granule preparation.

Formulation Example 2

A powder preparation for capsulation containing the followingingredients is prepared.

Ingredients Compound represented by the formula (1)  10 mg Lactose  79mg Corn Starch  10 mg Magnesium Stearate  1 mg 100 mg

A compound represented by the formula (1) and lactose are sifted througha 60-mesh sieve. Corn starch is sifted though a 120-mesh sieve. They aremixed with magnesium stearate in a V-type blender. The 10% powder is putin hard capsules No. 5, 100 mg each.

Formulation Example 3

A granule preparation for capsulation containing the followingingredients is prepared.

Ingredients Compound represented by the formula (1)  15 mg Lactose  90mg Corn Starch  42 mg HPC-L  3 mg 150 mg

A compound represented by the formula (1) and lactose are sifted througha 60-mesh sieve. Corn starch is sifted though a 120-mesh sieve. They aremixed in a V-type blender. The powder mixture is kneaded with alow-viscosity hydroxypropylcellulose (HPC-L) aqueous solution,granulated and dried. The resulting dry granules are sifted through ashaking sieve (12/60 mesh). The granules are put in hard capsules No. 4,150 mg each.

Formulation Example 4

A tablet preparation containing the following ingredients is prepared.

Ingredients Compound represented by the formula (1)  10 mg Lactose  90mg Microcrystalline cellulose  30 mg Magnesium Stearate  5 mg CMC-Na  15mg 150 mg

A compound represented by the formula (1), lactose, microcrystallinecellulose and CMC-Na (carboxymethylcellulose sodium salt) are siftedthrough a 60-mesh sieve and mixed. The powder mixture is mixed withmagnesium stearate to give a bulk powder mixture. The powder mixture iscompressed directly into 150 mg tablets.

Formulation Example 5

An intravenous preparation is prepared as follows.

Compound represented by the formula (1) 100 mg Saturated Fatty AcidGlyceride 1000 ml

Solutions having the above-mentioned composition are usuallyadministered to a patient intravenously at a rate of 1 ml per 1 minute.

INDUSTRIAL APPLICABILITY

The compounds of the present invention which have affinity forthrombopoietin receptor and act as thrombopoietin receptor agonists areuseful as preventive, therapeutic and improving agents for diseasesagainst which activation of the thrombopoietin receptor is effective,especially as drugs for hematological disorders accompanied by abnormalplatelet count and as drugs for diseases treated or prevented bystimulating differentiation and proliferation of vascular endothelialcells and endothelial progenitor cells, and are useful as medicines.

1. A method of increasing platelets in a human having a disease selectedfrom the group consisting of thrombocytopenia caused by bone marrowtransplantation, thrombocytopenia caused by surgery, thrombocytopeniacaused by infection, thrombocytopenia caused by gastrointestinalbleeding, aplastic anemia, idiopathic thrombocytopenic purpura,myelodysplastic syndrome, and thrombopoietin deficiency, the methodcomprising administering an isolated compound or its salt to the humanin need thereof in an amount sufficient to increase the platelets in thehuman in need thereof, wherein the isolated compound or its salt isrepresented by the formula (1) or its salt,

wherein each of R¹ and R³ is independently a hydrogen atom, SO₃H, a C₁₋₆alkyl group, a C₁₋₆ alkylcarbonyl group or a C₆₋₁₈ arylcarbonyl group,and wherein each of R², R⁴ and R^(a) is independently a hydrogen atom, ahydroxyl group or a C₁₋₆ alkoxy group; wherein the C₁₋₆ alkyl group, theC₁₋₆ alkylcarbonyl group and the C₆₋₁₈ arylcarbonyl group may beoptionally substituted with a halogen atom, a hydroxyl group, a C₂₋₆alkenyl group, a C₁₋₆ alkoxy group, a C₁₋₆ alkoxycarbonyl group, a C₆₋₁₈aryl group, a 2-pyridyl group, a 3-pyridyl group, a 4-pyridyl group, a2-furanyl group, a 3-furanyl group, a 2-thienyl group, a 3-thienyl groupor NR⁹R¹⁰, wherein the C₆₋₁₈ aryl group, the 2-pyridyl group, the3-pyridyl group, the 4-pyridyl group, the 2-furanyl group, the 3-furanylgroup, the 2-thienyl group and the 3-thienyl group may be optionallysubstituted with a halogen atom or a C₁₋₆ alkyl group, wherein each ofR⁹ and R¹⁰ is independently a hydrogen atom or a C₁₋₆ alkyl group or R⁹and R¹⁰ mean, together with each other, —(CH₂)_(n)X(CH₂)_(m)—, whereinthe C₁₋₆ alkyl group may be optionally substituted with a C₆₋₁₈ arylgroup wherein X is CR¹¹R¹², NR¹³, O or S, wherein each of R¹¹ and R¹² isindependently a hydrogen atom or a C₁₋₆ alkyl group, wherein R¹³ is ahydrogen atom or a C₁₋₆ alkyl group that may be optionally substitutedwith a C₆₋₁₈ aryl group, wherein n is 1, 2 or 3, and m is 1, 2 or 3,provided that n+m is 3, 4 or 5, wherein the C₁₋₆ alkyl group may beoptionally substituted with a C₆₋₁₈ aryl group, wherein R¹³ is ahydrogen atom or a C₁₋₆ alkyl group, and wherein the C₁₋₆ alkyl groupmay be optionally substituted with a C₆₋₁₈ aryl group.
 2. The method ofclaim 1, wherein in the isolated compound of or its salt that isadministered to the human, each of R¹ and R³ is independently a hydrogenatom, SO₃H, a C₁₋₆ alkyl group, a C₁₋₆ alkylcarbonyl group or a C₆₋₁₈arylcarbonyl group, and wherein the C₁₋₆ alkyl group, the C₁₋₆alkylcarbonyl group and the C₆₋₁₈ arylcarbonyl group may be optionallysubstituted with a hydroxyl group.
 3. The method of claim 1, wherein inthe isolated compound of or its salt that is administered to the human,each of R¹ and R³ is independently a hydrogen atom, SO₃H, a C₁₋₆ alkylgroup, a C₁₋₆ alkylcarbonyl group or a C₆₋₁₈ arylcarbonyl group, whereinthe C₁₋₆ alkyl group, the C₁₋₆ alkylcarbonyl group and the C₆₋₁₈arylcarbonyl group may be optionally substituted with NR⁹R¹⁰, whereineach of R⁹ and R¹⁰ is independently a hydrogen atom or a C₁₋₆ alkylgroup, or R⁹ and R¹⁰ mean, together with each other,—(CH₂)_(n)X(CH₂)_(m)—, wherein the C₁₋₆ alkyl group may be optionallysubstituted with a C₆₋₁₈ aryl group, wherein X is CR¹¹R¹², NR¹³, O or S,wherein n is 1, 2 or 3, and m is 1, 2 or 3, provided that n+m is 3, 4 or5, wherein each of R¹¹ and R¹² is independently a hydrogen atom or aC₁₋₆ alkyl group, wherein the C₁₋₆ alkyl group may be optionallysubstituted with a C₆₋₁₈ aryl group,  wherein R¹³ is a hydrogen atom ora C₁₋₆ alkyl group, and  wherein the C₁₋₆ alkyl group may be optionallysubstituted with a C₆₋₁₈ aryl group.
 4. The method of claim 1, whereinin the isolated compound of or its salt that is administered to thehuman, each of R¹ and R³ is independently a hydrogen atom or a C₁₋₆alkyl group.
 5. The method of claim 4, wherein in the isolated compoundor its salt that is administered to the human, each of R¹ and R³ isindependently a hydrogen atom or a methyl group, and each of R² and R⁴is independently a hydrogen atom, a hydroxyl group or a methoxy group.6. The method of claim 1, claim 2, claim 3, claim 4 or claim 5, whereinin the isolated compound or its salt that is administered to the human,R² is a hydrogen atom.
 7. The method of claim 6, wherein in the isolatedcompound or its salt that is administered to the human, each of R⁴ andR^(a) is independently a hydrogen atom or a methoxy group.
 8. The methodof claim 1, wherein the isolated compound or its salt that isadministered to the human in need thereof is administered as acomposition comprising the isolated compound of formula (1) or its saltand an excipient.
 9. The method of claim 1, wherein the compound or itssalt of formula (1) does not comprise a compound or its salt of formula(2):


10. A method of increasing platelets in a human, the method comprisingadministering an isolated compound or its salt to the human in an amountsufficient to increase the platelets in the human, wherein the isolatedcompound or its salt is represented by the formula (1) or its salt,

wherein each of R¹ and R³ is independently a hydrogen atom, SO₃H, a C₁₋₆alkyl group, a C₁₋₆ alkylcarbonyl group or a C₆₋₁₈ arylcarbonyl group,and wherein each of R², R⁴ and R^(a) is independently a hydrogen atom, ahydroxyl group or a C₁₋₆ alkoxy group; wherein the C₁₋₆ alkyl group, theC₁₋₆ alkylcarbonyl group and the C₆₋₁₈ arylcarbonyl group may beoptionally substituted with a halogen atom, a hydroxyl group, a C₂₋₆alkenyl group, a C₁₋₆ alkoxy group, a C₁₋₆ alkoxycarbonyl group, a C₆₋₁₈aryl group, a 2-pyridyl group, a 3-pyridyl group, a 4-pyridyl group, a2-furanyl group, a 3-furanyl group, a 2-thienyl group, a 3-thienyl groupor NR⁹R¹⁰, wherein the C₆₋₁₈ aryl group, the 2-pyridyl group, the3-pyridyl group, the 4-pyridyl group, the 2-furanyl group, the 3-furanylgroup, the 2-thienyl group and the 3-thienyl group may be optionallysubstituted with a halogen atom or a C₁₋₆ alkyl group, wherein each ofR⁹ and R¹⁰ is independently a hydrogen atom or a C₁₋₆ alkyl group or R⁹and R¹⁰ mean, together with each other, —(CH₂)_(n)X(CH₂)_(m)—, whereinthe C₁₋₆ alkyl group may be optionally substituted with a C₆₋₁₈ arylgroup wherein X is CR¹¹R¹², NR¹³, O or S, wherein each of R¹¹ and R¹² isindependently a hydrogen atom or a C₁₋₆ alkyl group, wherein R¹³ is ahydrogen atom or a C₁₋₆ alkyl group that may be optionally substitutedwith a C₆₋₁₈ aryl group, wherein n is 1, 2 or 3, and m is 1, 2 or 3,provided that n+m is 3, 4 or 5, wherein the C₁₋₆ alkyl group may beoptionally substituted with a C₆₋₁₈ aryl group, wherein R¹³ is ahydrogen atom or a C₁₋₆ alkyl group,  wherein the C₁₋₆ alkyl group maybe optionally substituted with a C₆₋₁₈ aryl group, with the proviso thatthe compound of formula (1) or its salt does not comprise a compound offormula (2) or its salt


11. The method of claim 10, wherein in the isolated compound of or itssalt that is administered to the human, each of R¹ and R³ isindependently a hydrogen atom, SO₃H, a C₁₋₆ alkyl group, a C₁₋₆alkylcarbonyl group or a C₆₋₁₈ arylcarbonyl group, wherein the C₁₋₆alkyl group, the C₁₋₆ alkylcarbonyl group and the C₆₋₁₈ arylcarbonylgroup may be optionally substituted with a hydroxyl group, with theproviso that the compound of formula (1) or its salt does not comprise acompound of formula (2) or its salt


12. The method of claim 10, wherein in the isolated compound of or itssalt that is administered to the human, each of R¹ and R³ isindependently a hydrogen atom, SO₃H, a C₁₋₆ alkyl group, a C₁₋₆alkylcarbonyl group or a C₆₋₁₈ arylcarbonyl group, wherein the C₁₋₆alkyl group, the C₁₋₆ alkylcarbonyl group and the C₆₋₁₈ arylcarbonylgroup may be optionally substituted with NR⁹R¹⁰, wherein each of R⁹ andR¹⁰ is independently a hydrogen atom or a C₁₋₆ alkyl group, or R⁹ andR¹⁰ mean, together with each other, —(CH₂)_(n)X(CH₂)_(m)—, wherein theC₁₋₆ alkyl group may be optionally substituted with a C₆₋₁₈ aryl group,wherein X is CR¹¹R¹², NR¹³, O or S, wherein n is 1, 2 or 3, and m is 1,2 or 3, provided that n+m is 3, 4 or 5, wherein each of R¹¹ and R¹² isindependently a hydrogen atom or a C₁₋₆ alkyl group, wherein the C₁₋₆alkyl group may be optionally substituted with a C₆₋₁₈ aryl group, wherein R¹³ is a hydrogen atom or a C₁₋₆ alkyl group,and  wherein theC₁₋₆ alkyl group may be optionally substituted with a C₆₋₁₈ aryl group,with the proviso that the compound of formula (1) or its salt does notcomprise a compound of formula (2) or its salt


13. The method of claim 10, wherein in the isolated compound of or itssalt that is administered to the human, each of R¹ and R³ isindependently a hydrogen atom or a C₁₋₆ alkyl group, with the provisothat the compound of formula (1) or its salt does not comprise acompound of formula (2) or its salt


14. The method of claim 13, wherein in the isolated compound or its saltthat is administered to the human, each of R¹ and R³ is independently ahydrogen atom or a methyl group, and each of R² and R⁴ is independentlya hydrogen atom, a hydroxyl group or a methoxy group, with the provisothat the compound of formula (1) or its salt does not comprise acompound of formula (2) or its salt


15. The method of claim 10, wherein in the isolated compound or its saltthat is administered to the human, R² is a hydrogen atom, with theproviso that the compound of formula (1) or its salt does not comprise acompound of formula (2) or its salt


16. The method of claim 11, wherein in the isolated compound or its saltthat is administered to the human, R² is a hydrogen atom, with theproviso that the compound of formula (1) or its salt does not comprise acompound of formula (2) or its salt


17. The method of claim 15, wherein in the isolated compound or its saltthat is administered to the human, each of R⁴ and R^(a) is independentlya hydrogen atom or a methoxy group, with the proviso that the compoundof formula (1) or its salt does not comprise a compound of formula (2)or its salt